Prestin, the motor protein of the outer hair cells in the organ of Corti in
the inner ear, plays a crucial role in the sensitivity of hearing due to its
role in mechano-sensoric transduction. Up to today, the molecular regulatory
mechanisms of prestin expression are still not completely understood. In this
study, mRNA expression of prestin and the transcription factors Pou4f3 (POU
domain, class 4, transcription factor 3), C/ebpβ (CCAAT/enhancer binding
protein beta), Sp1 (specificity protein 1), Carf (calcium response factor) and
Creb1 (cAMP response element binding protein) in rats were measured with the
help of Real-Time-PCR, applying different experimental conditions, and
referring to two reference systems. The total concentration of RNA in one
sample and the concentration of mRNA of the housekeeping gene Tbp were used as
independent reference systems. The expression of prestin and the transcription
factors investigated in this study were compared for: 1. the pattern of
expression; 2. the correlation between mRNA concentrations; and 3. the
existence of binding sites for the selected transcription factors in promoter
regions of prestin and of the transcription factors. The chosen conditions in
this study are known to have an impact on the expression of prestin. These are
firstly the changes during postnatal development in rats between the 2nd and
8th day. Secondly, expression changes were induced specifically in the
organotypic culture of the organ of Corti. For this purpose, the culture was
modified by adding thyroxin (T4), potassium chloride (KCl), and sodium
butyrate (SB), or retinoic acid (RA) respectively, in various concentrations.
In addition, the search was done for expression changes along the apical-basal
axis of the organ of Corti. This work shows the significant correlation
between mRNA expression of prestin and Pou4f in the postnatal development and
furthermore in the organotypic culture with the addition of thyroxin (0.5 µM),
sodium butyrate (up to 2000 µM), and increased potassium chloride (KCl)
concentrations (50 mM). Furthermore, both genes show an apical-basal gradient
along the apical-basal axis of the organ of Corti in the postnatal development
as well as in the 48h culture with the addition of thyroxin (0.5 mM). Another
significant correlation was demonstrated between the expression of C/ebp and
prestin in the postnatal development and in the 48h culture after adding
thyroxin (0.5 mM) and retinoic acid (up to 100 µM). In addition to that, C/ebp
also shows an apical-basal gradient in the organ of Corti in the postnatal
development. The expression of the transcription factor Carf correlates with
the prestin expression in the organotypic culture with the addition of
thyroxin (0.5 mM) as well as under the condition of applying KCL (50 mM).
There were no significant correlations between prestin expression and the
transcription factors Sp1 or Creb1a. Using a modified method of preparation,
an analysis was performed concerning local differences in the expression of
transcription factors in the inner ear between the area of hair cells and the
modiolus. It was found that the transcription factors Gata3 and C/ebpβ are
higher expressed in the organ of Corti than in the limbus. Pou4f3 is already
known to be expressed specifically in the external hair cells of the organ of
Corti. Compared to the modiolus, the expression of the transcription factors
Sp1, Carf, and Creb is not increased in the organ of Corti. An in silico
analysis of the promoter regions of prestin in human, rat, mouse, and bovine
provided evidence for the existence of homologous binding sites for Pou4f3,
C/ebpβ, and Carf in these regions. In addition to that, binding sites for
retinoic acid and thyroxin are found more frequently in the promoter sequences
of prestin rather than in random sequences with identical base composition.
These results indicate that the transcription factors Pou4f3, C/ebpβ and Carf
are potential candidates for the regulation of prestin expression. Further
experimental studies have to be performed to prove the functional relevance of
these transcription factors and their binding sites.